Coronary heart disease (CHD) continues to be a leading cause of mortality and morbidity worldwide. Age-related decline in mitochondrial function is a novel mechanism that may underlie multiple biological changes that increase the risk of atherosclerosis and CHD in the general population. We determined mtDNA copy number (mtDNA-CN) in whole blood, one of the primary relevant biological tissues for atherosclerosis, in 16,401 participants from 2 prospective community-based cohorts, the Atherosclerosis Risk in Communities (ARIC) study and the Cardiovascular Health Study (CHS). mtDNA-CN was significantly associated with age and was a robust predictor of overall mortality. The age-, sex-, and collection site-adjusted hazard ratio comparing mortality in the lowest to the highest quintiles of mtDNA-CN was 1.47 (P=4.24x10-14). Furthermore, lower mtDNA-CN at baseline was associated with an increased incidence of CHD (hazard ratio for a 1-SD decrease in mtDNA-CN 1.21, P=3.89x10-19) independent of traditional CHD risk factors. To establish the role of mtDNA-CN in CHD, we first propose to validate our findings in two additional longitudinal cohorts, the Multiethnic Study of Atherosclerosis (MESA) and the Rotterdam Study (RS), and together with ARIC and CHS, combine the rich clinical and genetic data available from these cohorts to comprehensively evaluate the role of mtDNA-CN in CHD, including determining whether mtDNA-CN is associated with subclinical atherosclerosis or with the development of traditional CHD risk factors. Second, we will use DNA from the baseline and 2 follow-up visits in ARIC to assess how longitudinal changes in mtDNA-CN influence future CHD risk. Third, we will conduct a GWAS, using both common and rare variants, to identify genetic determinants of mtDNA-CN in ~90,000 participants from ARIC, CHS, MESA, RS and additional CHARGE cohorts. Associated genetic variants will be used to determine whether decreased mtDNA-CN is causative for CHD using Mendelian randomization. Finally, we will functionally identify, characterize, and determine the downstream consequences of GWAS-identified genes that alter mtDNA-CN through a combination of mitochondrial functional assays, RNAseq, and computational network/pathway analysis. Computational analysis will identify shared patterns of dysregulation, interactions between regulatory genes, and pathways responsible for specific physiological changes. Given the preliminary data suggesting widespread phenotypic variation associated with altered mtDNA-CN, understanding the regulatory network associated with mtDNA-CN will be critical to elucidating the mechanisms that both regulate mtDNA-CN and/or drive the downstream phenotypic consequences. This proposal brings together unique expertise in GWAS, cardiovascular health, and mitochondrial function, and capitalizes on 4 well-established cohorts with ~5,800 cases of prevalent and incident CHD in over 20 years of follow-up, to elucidate the causal role of mtDNA-CN in CHD. More importantly, mtDNA-CN represents a potential novel etiology of CHD not captured by any established or proposed CHD risk factors.